Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/13—Plant traits

Definitions

• Sucrose storage organ of plants The present invention is in the field of industrial sugar production from crops and relates to the increase of the sucrose yield in the agricultural cultivation of crops.
• the invention relates to tonoplastoid proton / sugar antiporter proteins, in particular to tonoplastic proton / sucrose antiporter proteins, and the nucleic acids encoding them, and to their use for increasing the sucrose concentration of a sucrose storage organ of crops.
• Sugar is on the one hand a collective term for all sweet-tasting mono- and disaccharides, on the other hand, the common name for the disaccharide sucrose.
• Sucrose is the common household or granulated sugar and is also called sucrose.
• Sucrose is a dimer of one molecule each of ⁇ -D-glucose and ⁇ -D-fructose, which are linked to one another via an ⁇ , ⁇ -1,2-glycosidic bond.
• Sucrose is produced in plants by photosynthesis.
• the biosynthesis of sucrose takes place in the cytoplasm of the plant cells.
• the two triose phosphates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate which are obtained as a net gain in the carbon assimilation of photosynthesis (Calvin cycle)
• the monosaccharides UDP-glucose and fructose-6-phosphate are formed from the triose phosphates.
• fructose-1, 6-bisphosphate is first formed by a condensation reaction between glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
• Fructose-1, 6-bisphosphate is then converted by dephosphorylation to fructose-6-phosphate.
• Fructose-6-phosphate can be formed by isomerization and glucose-6-phosphate, which after prior Umisomerleiter to glucose-1-phosphate with uridine triphosphate (UTP) to uridine diphosphate-glucose (UDP-glucose). The subsequent condensation of UDP-glucose and fructose-6-phosphate to sucrose-6-phosphate is carried out by the enzyme sucrose-phosphate synthase
• Sucrose phosphate phosphatase cleaved so that sucrose is formed.
• Sucrose is a non-reducing disaccharide and therefore the most important transport sugar in plants.
• Sucrose is synthesized in the leaves of the plants and transported via the phloem into their storage organs, where it is accumulated in the vacuoles of the plant cells there as a nutrient and energy source.
• Sugar beets Beta vulgaris subsp. Vulgaris
• sugar cane Sacharum officinarum
• sugar palm Asrenga pinnata, Syn. Arenga saccharifera Labil., Primarily in Indonesia
• the sugarcane contains sugars - predominantly sucrose - with a share of usually 10 to 20% in the marrow of the plant (the
• the cane sugar is made by crystallization and
• the sugar beet is a biennial plant, which in the first year creates a sugar supply in the beet body, which serves as food in the second year of the flowering plant.
• the sugar is usually made from chips of sugar beet in an extraction process with water.
• the extract can then be treated with calcium oxide to the plant acids such as oxalic or tartaric acid and the
• sucrose concentration of the beet body is about 15 to 20% by weight, based on the fresh weight of the beet body.
• the international application published under WO 2010/072210 A1 discloses a process for increasing the sucrose yield in the agricultural cultivation of sugar beet.
• the method uses sugar beet or cane plants whose genetic makeup is based on
• a nucleic acid which is suitable in a plant cell for reducing the enzymatic activity of an invertase, to form a
• Sucrose concentration is increased compared to the sucrose concentration of a non-modified Kontrollsaccharosepeicherorgans the same genotype in a comparable developmental stage.
• Vegetable vacuoles play a central role in the long- or short-term storage of sugars, because the vacuole occupies a volume of about 90% as an organelle in a photosynthetically active plant cell (Martinola, E. et al. (2007) "Vacuolar transporters and their essential role in plant metabolism ", J. Exp. Bot 58: 83-102). Vacuoles are, therefore, due to their size for the Storage of sugars of immense importance (Neuhaus, HE (2007) "Transport of primary metabolites across the plant vacuolar membrane", FEBS Lett.
• storage tissues such as the taproot of the sugar beet (Beta vulgaris) and the marrow of the sugar cane (Saccharum officinarum) accumulate large amounts of sucrose in the vacuoles of the cells of their storage organs in order to use them as an energy source for their plant metabolism can.
• TMT tonoplastidäre monosaccharide transporter
• Transport protein is located in the membrane of the vacuole, the tonoplast.
• the monoclonal monosaccharide transporter (TMT) protein comprises three isoforms in Arabidopsis thaliana, designated AfTMV1, AfTMT2, and ⁇ " MT3.”
• the genes for AHMT * ! And> 4fTMT2 have a tissue- and cell-type specific expression pattern, whereas It was shown via TMT gene knockouts that the plants thus modified accumulated significantly less glucose and fructose in their vacuoles compared to wild-type plants
• the Arabidopsis thaliana TMT1 monoclonal tonoplastoid monosaccharide transporter has been shown to be electrophysiologically proton-driven
• Sucrose antiporter characterizes the glucose and sucrose with The same type of specificity is transported across the vacuolar membrane (Schulz, A. et al. (2011) “Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1 / 2", The Plant Journal 68: 129-136) the same article is also the sucrose transport protein SUC4 of
• Arabidopsis thaliana is characterized as a proton / sucrose symporter, which should also be located in the vacuolar membrane.
• Sucrose in a storage organ is not disclosed.
• the object of the present invention was based on the identification of the proteins responsible for the import of sugar into the vacuole of the sugar beet root cell,
• the invention relates to a nucleic acid molecule which encodes a protoplast / sugar antiporter tonoplastoid protein.
• the nucleic acid molecule encodes a protoplast / sugar antiporter tonoplastoid protein which is specific for sucrose.
• proton / sugar antiporter protein which is specific for sucrose is also referred to as proton / sucrose antiporter protein.
• the invention relates to a recombinant gene comprising a nucleic acid molecule according to the first aspect or a
• the nucleic acid molecule may be operably linked to at least one regulatory element.
• the invention relates to a vector or a mobile genetic element comprising a nucleic acid molecule according to the first aspect or a recombinant gene according to the second aspect.
• the invention relates to a eukaryotic
• a host cell or a prokaryotic host cell comprising a nucleic acid molecule according to the first aspect, preferably as a transgene, a recombinant gene according to the second aspect or a vector or mobile genetic element according to the third aspect.
• the invention relates to a protein which is known as
• tonoplastide proton / sugar antiporter which is preferably specific for sucrose, or preferably as tonoplastidärer
• the invention relates to a transgenic plant cell which comprises a nucleic acid molecule according to the first aspect as a transgene, a recombinant gene according to the second aspect as a transgene or a vector or mobile genetic element according to the third aspect, and a transgenic plant or parts thereof that at least one such trangene
• the invention relates to seeds of a transgenic plant according to the preceding aspect, wherein the seed is a
• Nucleic acid molecule according to the first aspect as a transgene, a recombinant gene according to the second aspect as a transgene or a vector or mobile genetic element according to the third aspect.
• the invention relates to methods of producing transgenic plants.
• the invention relates to methods of increasing the sucrose concentration of a sucrose storage organ of a plant. In another aspect, the invention relates to methods for identifying a plant capable of producing an increased sucrose concentration in a sucrose storage organ of the plant.
• the invention relates to oligonucleotides suitable for use as molecular markers for the diagnostic detection of a nucleic acid molecule according to the first aspect.
• the invention relates to antibodies which are diagnostic for a protein which functions as a tonoplastoid proton / sugar antiporter, which is preferably specific for sucrose, or preferably as a tonoplastic proton / sucrose antiporter.
• the invention relates to the use of tonoplastoid proton / sugar antiporter proteins to increase the
• sucrose concentration of a sucrose storage organ of a plant is a sucrose concentration of a sucrose storage organ of a plant.
• FIG 1 is a table showing the identities and similarities of the amino acid sequences of three Arabidopsis thaliana paralograft monopolysaccharide transporter (TMT) proteins with the four paralogous tonoplastoid sugar transporter (TST) proteins from Beta vulgaris
• TMT Arabidopsis thaliana paralograft monopolysaccharide transporter
• TST paralogous tonoplastoid sugar transporter
• FIG. 2 shows a cladogram in which the phylogenetic relationships of the three paralogous mononosaccharide transporter (TMT) -totaloid proteins of
• FIG. 3 shows a bar chart showing the sucrose concentration in FIG.
• Figure 4 is a bar graph showing the relative mRNA levels of the four paralogous TST genes of Beta vulgaris at two different levels
• Figure 5 is a bar graph showing the concentration of various sugars in the leaves of the sugar beet "Belladonna KWS"
• FIG. 6 shows a bar graph from which the relative mRNA quantity for the four paralogue ⁇ vTST genes in leaves of the sugar beet "Belladonna KWS" emerge at different development times.
• Figure 7 is a bar chart showing that of different sugars
• ⁇ vTST2.1 the protein referred to herein as ⁇ vTST2.1 as one of the most abundant proteins present in the vacuolar membrane of taproot sugarcane cells, and surprisingly
• the protein ⁇ vTST2.1 as a tonoplastidic sugar transporter can specifically import sucrose from the cytosol into the vacuoles of plant cells. Therefore put this protein as well as proteins with same
• the invention according to the first aspect relates to nucleic acid molecules which encode a protoplast / sugar antiporter tonoplastoid protein, preferably a proton / sucrose antiporter tonoplastoid protein.
• the nucleic acid molecule encoding a protoplast / sucrose antiporter tonoplastoid protein comprises
• Nucleic acid molecule selected from the group:
• nucleic acid molecule having a nucleotide sequence according to SEQ ID NO: 2 or a nucleic acid molecule having a nucleotide sequence which has an identity of at least 80% to the nucleotide sequence according to SEQ ID NO: 2; b) a nucleic acid molecule having a nucleotide sequence which is complementary to one of the nucleotide sequences according to a);
• nucleic acid molecule which hybridizes with a nucleic acid molecule according to a) or b);
• nucleic acid molecule having a nucleotide sequence which encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 1, or a
• nucleic acid molecule having a nucleotide sequence which codes for a homologue, an analogue or an orthologue of the polypeptide according to SEQ ID NO: 1.
• nucleic acid molecule encoding a protoplast / sugar antiporter tonoplastoid protein comprises
• Nucleic acid molecule selected from the group:
• nucleic acid molecule having a nucleotide sequence which is complementary to one of the nucleotide sequences according to a);
• nucleic acid molecule which hybridizes with a nucleic acid molecule according to a) or b);
• nucleic acid molecule having a nucleotide sequence which codes for a homologue, an analogue or an orthologue of the polypeptide according to SEQ ID NO: 3, 5 or 7.
• nucleic acid molecule having a nucleotide sequence comprises not only nucleic acid molecules whose nucleotide sequence consists of the nucleotide sequence then described in more detail, but also nucleic acid molecules which additionally have at least one nucleotide or nucleotide sequences for the nucleotide sequence which is then specified.
• the nucleic acid molecule encodes an amino acid sequence according to SEQ ID NO: 1, 3, 5 or 7.
• the nucleic acid molecule can also encode an amino acid sequence in which at least one amino acid residue of the amino acid sequence is replaced by an amino acid which is similar has chemical properties,
• amino acid substitution is replaced (conservative or semiconservative amino acid exchange).
• conservative amino acid substitution one amino acid is replaced by another amino acid with similar chemical properties.
• semiconservative amino acid substitution one amino acid is replaced by another amino acid of similar steric conformation.
• the exchange preferably has no consequences for protein function. Examples of amino acid substitutions are Asp and Glu, Leu and Ile, Ala and Val, Arg and Lys, and Phe and Trp.
• the nucleotide sequences of the nucleic acids and / or the amino acid sequences which are encoded by the nucleotide sequences have an identity of at least 80%, at least 85%, preferably at least 90%, particularly preferably at least 95%, at least 96%, at least 97% or at least 98%, and most preferably of at least 99% to the nucleotide sequence according to SEQ ID NO: 2, 4, 6 or 8 or the amino acid sequence according to SEQ ID NO: 1, 3, 5 or 7 on.
• hybridize means to hybridize under standard conditions as described in Sambrook et al. (1989) "Molecular Cloning, A Laboratory Manual” (Cold Spring Harbor Laboratory Press, New York), preferably under stringent conditions.
• Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C followed by multiple washes in 0.1 x SSC at 65 ° C for a total of about 1 hour.
• hybridization in 4 x SSC at 37 ° C followed by multiple washes in 1 x SSC is less stringent hybridization conditions
• “Stringent hybridization conditions” may also mean: hybridizing at 68 ° C in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and then washing twice with 2 x SSC and 0, 1% SDS at 68 ° C.
• sucrose For the purposes of the invention is under "specific for sucrose” or “highly specific for sucrose” or “sucrose-specific transport” or “sucrose highly specific transport” or “specificity for sucrose” or Sucrose specificity "means that the specificity of a proton / sugar antiporter tonoplastide protein for sucrose versus another sugar is at least 5-fold, 10-fold or 15-fold, preferably at least 18-fold, 20-fold, 22-fold, 24-fold, 26-fold or 28-fold, more preferably at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold or at least 39-fold, and most preferably at least 40-fold higher Furthermore, this may also mean that the specificity of a protoplast / sugar antiporter tonoplastiddidul protein for sucrose against a monosaccharide such as glucose or fructose at least 5-
• a “homologue” is understood to mean a protein of the same phylogenetic origin, an “analog” to mean a protein which has the same function, but a different one
• phylogenetic origin and by an "orthologue" means a protein from another species that performs the same function.
• the invention relates to a recombinant gene comprising a nucleic acid molecule according to the first aspect or a
• Nucleic acid molecule having a nucleotide sequence which preferably encodes a protoplast / sucrose antiporter tonoplastoid protein.
• Nucleic acid molecule may be operably linked to at least one regulatory element.
• regulatory element nucleotide sequences that are not part of the protein-encoding nucleotide sequence but that mediate expression of the protein-encoding nucleotide sequence
• promoters cis-regulatory elements, enhancers, introns or terminators.
• cis-regulatory elements e.g., promoters, cis-regulatory elements, enhancers, introns or terminators.
• the regulatory elements are functional in a living plant cell.
• operatively linked means that a regulatory element is linked to the protein-encoding nucleotide sequence in such a way, ie is positioned to the protein-encoding nucleotide sequence on, for example, a nucleic acid molecule such that expression of the protein-encoding nucleotide sequence under control of the regulatory element in a living cell can be done.
• a “promoter” is a nucleotide sequence which regulates the expression of a gene, which is usually located at the 5 'end of a gene and mediates, via interaction with certain DNA-binding proteins, the start of transcription by RNA polymerase
• Promoters that are functional in plant cells include constitutive promoters such as viral promoters, for example the CaM35S promoter, a double CaM35S promoter, or as plant promoters, for example ubiquitin promoters as described in EP 0 305 668 and US 6,528,701.
• constitutive promoters such as viral promoters, for example the CaM35S promoter, a double CaM35S promoter, or as plant promoters, for example ubiquitin promoters as described in EP 0 305 668 and US 6,528,701.
• promoters which have, for example, specific activity at specific development stages or inducibility by environmental factors such as biotic or abiotic stress or a
• those promoters can be used which show an increased specificity for the sucrose storage organ or parts thereof, ie are active in particular in this sucrose storage organ or in parts thereof.
• the promoter can be, for example, a root-specific or taproot-specific promoter.
• WO 02/40687 Oltmanns, H. et al. (2006) "Taproot promoters cause tissue specific gene expression within the storage root of sugar beet", Planta 224: 485-495, Noh, Seol Ah, et al.
• Halm-specific promoters may be used, such as for example, Goshu Abraha, Tsion. "Isolation and characterization of a culm-specific promoter element from sugarcane", Diss. Arthurosch: University of Whybosch, 2005; Govender, C. "Stern specific promoters from sorghum and maize for use in sugarcane.” Diss. Arthurosch: Whybosch University, 2008; and Mudge, SR et al. (2013) "Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane ", Plant Biotechnology Journal 1: 502-509).
• Synthetic promoters have also been found to be suitable promoters. These are promoters created by molecular biology techniques that are not found in nature in this embodiment.
• a synthetic promoter is a minimalistic promoter which, in addition to anal promoter, contains only one or more selected, defined cis elements. These cis elements are binding sites for DNA binding proteins such as transcription factors and are derived from natural
• the "minimal promoter” or “core” promoter is a nucleotide sequence that contains the binding sites for the basal transcription factor complex and allows the accurate initiation of transcription by RNA polymerase II. Characteristic sequence motifs of the minimal promoter are the TATA box, the initiator element (Inr), the TFBII recognition element (BRE) and the downstream core promoter element (OPE) individually or in combination. Available is the minimal promoter or its sequence motifs, for example, from any plant, bacterial, fungal or viral gene.
• Cross elements are nucleotide sequences that reside on the same nucleic acid molecule as the protein-encoding nucleotide sequence that is to be expressed. "Cis elements need not encode RNA or protein and can be expressed in
• expressing protein-encoding nucleotide sequence are often necessary binding motifs for in particular transcription factors, which here as trans-acting elements (from Latin trans beyond '), seen from a molecular point of view, intervene in the regulation of the transcription of this gene. If cis elements lead to inhibition of transcription, they are called silencers. Cis elements that lead to an increase in transcription, be
• Called enhancer The totality of the cis / trans activities in the promoter ultimately determines the intensity with which the RNA polymerase performs the transcription.
• a promoter may also be a chimeric promoter and / or a promoter modified by cis elements.
• the modification of a promoter can also mean the additional introduction of an ice element into the promoter, which for example
• the modification also includes a multimerization of a cis element, in particular a
• Such a modified promoter may have altered properties in terms of, for example, specificity, level of expression, or in comparison to the native version
• Terminators are nucleotide sequences on the DNA that usually label the end of a gene and lead to the termination of transcription.
• the nucleotide sequence coding for the proton / sugar antiporter tonoplastoid protein, in particular the nucleotide sequence coding for the protoplast / sucrose antiporter protein, and the nucleotide sequence of the at least one regulatory element are heterologous. This means that they come from different species or do not naturally occur in the intended combination in a species.
• the invention relates to a vector or a mobile genetic element comprising a nucleic acid molecule having a
• a vector is understood to mean a transport vehicle for a nucleic acid molecule according to the first aspect or a recombinant gene according to the second aspect, in particular for the transfer of a foreign nucleic acid into a living recipient cell.
• the living recipient cell may be a eukaryotic cell or a prokaryotic cell.
• the vectors include, for example, plasmids, cosmids, artificial
• YACs Yeast chromosomes
• BACs artificial bacterial chromosomes
• PACs artificial P1 chromosomes
• modified viruses such as adenoviruses, retroviruses and phages.
• Mobile genetic elements are nucleotide sequences whose position in the genome of an organism is variable. For example, self-interested nucleotide sequences such as transposons belong to the mobile genetic elements.
• Retroelements insertion sequences and inteins, but also group II introns, inserting plasmids and some bacteriophages such as Mu phage.
• the invention relates to a eukaryotic
• a host cell or prokaryotic host cell comprising a nucleic acid molecule according to the first aspect as a transgene, a recombinant gene according to the second aspect as a transgene or a vector or a mobile genetic element according to the third aspect as a transgene. That means that Nucleic acid molecule, the recombinant gene and / or the vector or the mobile genetic element has been introduced into the host cell, for example by means of transformation or transfection.
• Host cells are bacteria of the genus A. tumefaciens, E. coli and B. subtilis.
• Examples of eukaryotic host cells are yeast cells such as Saccharomyces or Schizosaccharomyces, but also cells of animal or plant origin.
• the invention relates to proteins which are known as
• tonoplastide proton / sucrose antiporter work.
• This antiporter is specific for sucrose.
• the protein is replaced by a
• the proton / sucrose antiporter tonoplastoid protein is selected from the group of proteins which
• a) have an amino acid sequence according to SEQ ID NO: 1;
• b) have an amino acid sequence which has an identity of at least 80% to the amino acid sequence according to SEQ ID NO: 1;
• Transporter Protein 2 from Arabidopsis thaliana ( ⁇ " ⁇ 2) The identity of these two amino acid sequences is 68% and they are under
• the invention relates to proteins that function as a tonoplastoid proton / sugar antiporter.
• the protein is encoded by a nucleic acid molecule according to the first aspect.
• the protoplast / sugar antiporter tonoplastoid protein is selected from the group of proteins which
• a) have an amino acid sequence according to SEQ ID NO: 3, 5 or 7;
• b) have an amino acid sequence which has an identity of at least 80% to the amino acid sequence according to SEQ ID NO: 3, 5 or 7;
• the tonoplastoid proton / sugar antiporter protein according to SEQ ID NO: 3 is also referred to as 8t TST1, according to SEQ ID NO: 5 also as ßi / TST2.2 and according to SEQ ID NO: 7 as 8 TST3.
• Antiporter protein ß TST2.1 as well as the other tonoplastidDS
• the invention relates to a transgenic plant cell which comprises a nucleic acid molecule according to the first aspect as transgene, a recombinant gene according to the second aspect as transgene or a vector or a transgenic plant or parts thereof comprising at least one such plant cell.
• the transgenic plant or parts thereof also include a nucleic acid molecule according to the first aspect
• Transgene a recombinant gene according to the second aspect as a transgene or a vector or mobile genetic element according to the third aspect as a transgene.
• the invention relates to seeds of a transgenic plant according to the preceding aspect, wherein the seed, and in particular at least one embryonic cell of the seed, a nucleic acid molecule according to the first aspect as transgene, a recombinant gene according to the second aspect as transgene or a vector or mobile genetic element according to the third aspect.
• the plant cell is the cell of a monocotyledonous plant. In another embodiment, the plant cell is a cell of a dicotyledonous plant. According to a further and / or additional embodiment, it is in the
• the transgenic plant is selected from the group consisting of Beta vulgaris, Saccharum officinarum, Arenga saccharifera, Acer saccharum and Sorghum sp. includes.
• the parts of a transgenic plant or the seed of a transgenic plant are from the group of plants which contain Beta vulgaris, Saccharum officinarum, Arenga saccharifera, Acer saccharum and Sorghum sp. includes.
• the transgenic plant cell, the transgenic plant or the parts of the transgenic plant which is preferably the sucrose storage organ of the plant, a higher sucrose concentration than the isogenic plant cell or plant cultured under identical conditions.
• parts of a plant may be associated with or separated from the whole intact plant. Such parts include, for example, organs, tissues, cells and seeds of the plant.
• the higher sucrose concentration is based on a higher sucrose concentration in the plant vacuole, in particular in the vacuole of at least one cell of the sucrose storage organ of the plant.
• a plant with a higher sucrose concentration is particularly preferred.
• sucrose also increased sucrose yield.
• yield means the yield of sucrose from the sucrose storage organ with respect to a defined cultivation area (for example one hectare) or with reference to the weight of a sucrose storage organ, taking into account the water content in the sucrose storage organ (preferably normalizing to fresh weight or dry weight).
• the invention relates to a process for the production of transgenic plants, the process comprising at least the following steps:
• step (b) regenerating the transgenic plant from the plant cell obtained in step (a).
• the transgenic plant resulting from the process is capable of concentrating sucrose in the vacuoles of its cells, preferably in the vacuoles of the cells of its sucrose storage organ, higher than an isogenic control plant cultured under identical conditions.
• Control plants "or” isogenic plant cells “means those plants or plant cells which have been used as starting material for the production of transgenic plants or transgenic plant cells.
• the genome of the transgenic plants and / or plant cells insofar as they are genetically modified plants or plant cells, do not differ from each other except for the genetically transmitted genes and / or introduced nucleotide sequences.
• the transgenic plant expresses or overexpresses the nucleotide sequence coding for at least one proton / sugar antiporter protein in at least one cell.
• nucleic acid molecule for example by way of transformation, can be carried out by techniques that are known in the art to a person skilled in the art.
• the nucleic acid molecule can be obtained by infecting a plant tissue or a plant cell with Agrobacterium tumefaciens containing the nucleic acid sequence to be transferred on its in the
• nucleic acid to be introduced into the plant cell is applied to gold particles or tungsten particles, which are then shot into the cells at high speed.
• protoplast transformation in which either polyethylene glycol is added to the protoplasts in the presence of the nucleic acid molecules to be introduced or the protoplasts are exposed to a short current impulse so that the protoplast membrane becomes transiently permeable to the nucleic acid molecules .
• the nucleic acid molecule according to the first aspect, the recombinant gene according to the second aspect and / or the vector or the mobile genetic element according to the third aspect are stably introduced into the genome of the cell of the plant. That means the transferred
• Nucleic acid sequence can be stably inherited after regeneration of a plant from this to a descendant plant.
• the transgenicity of the plants can be checked by polymerase chain reaction using suitable oligonucleotide primers. After regeneration, the transformants can be grown and selfed to obtain seed in the greenhouse.
• the ones to be transformed are
• the plant cells to be transformed are cells of dicotyledonous plants. According to a further and / or additional embodiment, the plant cells to be transformed are cells of a plant selected from the group of species or higher
• Genes selected, the beta vulgaris, Saccharum officinarum, Arenga saccharifera, Acer saccharum and Sorghum sp. includes.
• the invention relates to methods for increasing the sucrose concentration of a sucrose storage organ of a plant by expression or overexpression of a proton / sugar tonoplastoid
• Antiporter protein in particular a tonoplastoid proton / sucrose antiporter protein, in at least one cell of the plant.
• Expression or overexpression may be by genetic modification of at least one cell of the plant and comprises (1) the introduction of a nucleic acid molecule according to the first aspect, a recombinant gene according to the second aspect and / or a vector or mobile genetic element according to the third aspect into at least one cell of a plant, whereby an additional expression or an overexpression of a tonoplastidDS protons / Sugar antiporter protein is effected, or
• an endogenous regulatory element such as a promoter, that regulates the expression of an endogenous gene encoding a protoplast / sugar antiporter tonoplastoid protein
• Proton / sugar antiporter protein especially a tonoplastoid
• Proton / sucrose antiporter protein in at least one cell of the plant, the import of sucrose into the vacuoles of the genetically modified cell is improved. This also increases the sucrose concentration in the vacuoles of this cell as compared to an isogenic plant cell.
• Sucrose concentration "or a" higher sucrose concentration of a sucrose storage organ of a plant "means an increase of the average sucrose concentration, based on the fresh weight of the sucrose storage organ, compared to a non-transgenic (isogenic) control plant cultured under identical conditions of at least 0.2%, 0, 4%, 0.6%, 0.8% or 1%, preferably of at least 1, 2%, 1, 4%, 1, 6%, 1, 8% or 2%, more preferably of at least 2.5% , 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, or 10%, and most preferably at least 15%.
• the term “overexpressed” is understood to mean that the amount of protoplast / sugar antiporter protein in a proton Plant, plant cell or their Tonoplasten is higher than in the isogenic plant, isogenic plant cell or their Tonoplasten.
• Sucrose concentration of a sucrose storage organ of a plant the expression and / or overexpression of the nucleotide sequence of a nucleic acid molecule coding for a protoplast / sugar antiporter tonoplastoid protein of a nucleic acid molecule according to the first aspect of the invention.
• transgenic plant is prepared according to the method described above, wherein the expression and / or
• a construct comprising a strong promoter and a nucleotide sequence according to the first aspect of the invention can also be introduced into a plant cell to be transformed.
• Proton / sucrose-antiporter protein-encoding gene modified to be more active in the transgenic plant than in the isogenic control plant.
• Means for modifying an endogenous promoter may be, for example, TALENs or zinc finger nucleases.
• additional gene copies of the endogenous gene encoding a protoplast / sugar antiporter tonoplastoid protein in particular the endogenous gene encoding a proton / sucrose antiporter tonoplastoid protein, including its natural promoter, may be incorporated in the
• Plant cell are introduced.
• the proton / sucrose antiporter tonoplastoid protein is selected from the group consisting of
• the invention relates to methods of identifying a plant capable of producing an increased sucrose concentration in its sucrose storage gene.
• the plants to be identified may be subjected to marker assisted identification.
• the DNA of each plant to be examined is isolated and subjected to either a polymerase chain reaction (PCR) with suitable oligonucleotide primers, so that from the analysis of the reaction products of the PCR, either via gel chromatography or fluorescence detection as in RT-PCR, those plants are identified which, by virtue of their genetic make-up, are capable of producing an increased sucrose concentration in their sucrose storage logan.
• PCR polymerase chain reaction
• the genetic makeup of the plants to be identified can also be carried out by means of a restriction length polymorphism in which the isolated DNA is hydrolyzed with various Restitechnischsendonukleasen, the
• Suitable exemplary oligonucleotides for identification of transgenic plants which are suitable for generating an increased sucrose concentration in their sucrose storage organ because they express the nucleotide sequence according to SEQ ID NO: 2 or
• oligonucleotides comprising SEQ ID NO: 15 to SEQ ID NO: 26.
• the person skilled in the art is aware of how he can also provide suitable oligonucleotides for homologs, analogs or orthologs of SEQ ID NO: 2.
• the identification of the plants which are capable of producing an increased sucrose concentration in their sucrose storage logan is effected not by their genetic makeup but by the expression of their proton / sucrose antiporter tonoplastoid proteins. This can be done, for example, at the level of mRNA by measuring the amount of mRNA for the tonoplastidDS Proton / deoxyribonucleotide sequences encoding sugar antiporter proteins, in particular the deoxyribonucleotide sequences coding for the proton / sucrose antiporter protoplasmic proteins;
• Identification of plants capable of producing an increased sucrose concentration in their sucrose storage logan may also be achieved by quantitative detection of the amount of proton proton / sugar antiporter protein, in particular on proton proton / sucrose antiporter protein, in done a plant part.
• a so-called Western Blot is used, in which the electrophoretically separated proteins of the plant part, preferably the vacuoles, particularly preferably the
• Vacuole membrane of this part with one for one or more above
• tonoplastidäre proton / sugar-antiporter proteins specific antibodies are incubated.
• a secondary antibody which binds the antibody specific for one or more proton / sugar antiporter proteins described above and which has a detectable label
• the amount of proton proton / sugar antiporter protein, in particular on tonoplast protons may be increased.
• Sucrose antiporter protein in which plant part is determined and the plants identified which are capable of producing an increased sucrose concentration in their sucrose storage logan.
• the present invention thus also extends to the plants identified by the aforementioned method which are capable of increasing
• the invention relates to oligonucleotides suitable for use as molecular markers for the diagnostic detection of a nucleic acid molecule according to the first aspect.
• Oligonucleotides selected from the group comprising the oligonucleotides according to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. These can be used as molecular markers for the diagnostic detection of a nucleic acid molecule having a nucleotide sequence according to SEQ ID NO: 2.
• the invention relates to antibodies that are diagnostic of a protein useful as a protoplast / sugar antiporter tonoplastide,
• the diagnostic antibody is a monoclonal antibody. In an alternative embodiment, the diagnostic antibody is part of a polyclonal antiserum.
• the diagnostic antibody or polyclonal antiserum is specific for a particular protoplast / sugar antiporter tonoplastoid protein such as tonoplastide proton / sucrose antiporter protein.
• the diagnostic antibody recognizes and binds an epitope on the loop between the sixth and seventh transmembrane domains of a proton / sucrose antiporter protein.
• the invention relates to the use of a proton / sugar antiporter tonoplastoid protein to increase the
• sucrose concentration of a sucrose storage organ of a plant is a sucrose concentration of a sucrose storage organ of a plant.
• the use of a proton / sugar antiporter tonoplastoid protein to increase the sucrose concentration of a sucrose storage organ of a plant comprises increasing the
• nucleic acid molecule encoding the protoplast / sugar antiporter tonoplastoid protein.
• the nucleic acid molecule comprises i. a nucleic acid molecule having a nucleotide sequence according to SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, or having a nucleotide sequence which has an identity of at least 80% to one of the nucleotide sequences according to SEQ ID NO: 2, 4, 6 , 8, 10, 12 or 14;
• nucleic acid molecule having a nucleotide sequence which is complementary to one of the nucleotide sequences according to i. is;
• nucleic acid molecules conjugated to any of the nucleic acid molecules according to i. or ii. hybridized;
• nucleic acid molecule which encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 or which encodes a polypeptide having an amino acid sequence which has an identity of at least 80% to one of the amino acid sequences according to SEQ ID NO: 1, 3, 5, 7, 9, 11 or
• the nucleic acid molecule according to SEQ ID NO: 2 encodes the tonoplastoid proton / sugar antiporter TST2.1 of Beta vulgaris having the amino acid sequence according to SEQ ID NO: 1.
• the nucleic acid molecule according to SEQ ID NO: 4 encodes the beta-proton tonoplastoid proton / sugar antiporter TST1 having the amino acid sequence according to SEQ ID NO: 3.
• the nucleic acid molecule according to SEQ ID NO: 6 codes for the tonoplastoid proton / sugar antiporter TST2.2 of beta vulgaris having the amino acid sequence according to SEQ ID NO: 5.
• the nucleic acid molecule according to SEQ ID NO: 8 encodes the beta-progesterone proton / sugar antiporter TST3 of beta vulgaris having the amino acid sequence according to SEQ ID NO: 7.
• the nucleic acid molecule according to SEQ ID NO: 10 encodes the protoplast / sugar antiporter TMT1 of tonoplastoid protease Arabidopsis thaliana with the
• the nucleic acid molecule according to SEQ ID NO: 12 codes for the proton / sugar antiporter TMT2 of Arabidopsis thaliana with the
• the nucleic acid molecule according to SEQ ID NO: 14 codes for the proton / sugar antiporter TMT3 of Arabidopsis thaliana with the
• the amount of proton / sugar antiporter protein in the vacuolar membrane of this plant can be increased, in particular in the membranes of Vakuolen the sucrose storage organs of this plant, so that more sucrose in the Vacuoles of the plant
• Saccharose noma the plant, compared with an isogenic control plant cultured under identical conditions, is increased.
• sucrose yield per plant, per Saccharose Proforgan and / or per acreage can be increased.
• TST2.1 of Beta vulgaris is the tonoplastidäre membrane protein, which can import as a proton / sugar antiporter highly specific sucrose into the vacuole of a plant cell.
• sugar beets of the varieties “Belladonna KWS” and “Brigadier” were used.
• the seeds for the variety “Belladonna KWS” were provided by KWS Saat AG, Einbeck, DE, and the seeds for "Brigadier” beets were purchased in the local seed trade.
• plants and plant cells of Nicotiana benthamiana and Arabidopsis thaliana were used.
• the plants grew in growth chambers on the standard substrate ED-73 of the company Méserde- and Humuswerke Gebr. Patzer GmbH & Co. KG in a light-dark cycle of 10 hours light and 14 hours of darkness, 22 ° C and 125 pmol quanta m " 2 s "1 .
• Attstl -2 T-DNA double gene knockout mutant has been described in the prior art (Wormit, A. et al. (2006) Molecular Identification and Physiological Characterization of a Novel Monosaccharide Transporter from
• the taproot tissue of the sugar beet was harvested with a vegetable slicer, immediately frozen in liquid nitrogen and until quantitative
• Vacuoles were prepared by the method of Leigh and Branton (Leigh, RA and Branton, D. (1976) "Isolation of Vacuoles from Root Storage Tissue of Beta Vulgaris", L. Plant Physiol. 58: 656-662) with the following changes
• the taproot was cut with a vegetable slicer into slices of 0.1 to 0.2 mm in thickness, which were immediately placed in a collecting medium (1 M
• Sorbitol, 1mM DTT, 5mM EDTA, 50mM Tris-HCl, pH, 7.6) at room temperature were incubated. Subsequently, the thin slices of the taproot tissue were minced with a razor blade in the collection medium (1 M sorbitol, 1 mM DTT, 5 mM EDTA, 50 mM Tris-HCl, pH 7.6)
• Example 5 Liquid chromatography and tandem mass spectrometry
• the sediments of isolated tonoplast membranes from 2 or 5 month old plants were taken up in buffer (4% SDS, 50 mM NH4HCO3) at a concentration of 1 pg / ml.
• the ingested proteins were precipitated overnight at -20 ° C in 80% acetone and further processed as from Mühlhaus
• cDNA Complementary DNA of Beta vulgaris was prepared by reverse transcription of RNA isolated from taproots or leaves. All polymerase chain reactions (PCR) were performed with the Phusion-HF DNA polymerase (Thermo Scientific).
• the pUBQ: ⁇ vTST1-GFP fusion construct was constructed using the pUBC-GFP-Dest vector (Grefen et al. (2010) "Aubiquitin-10 promoter-based vector set for fluorescent protein tagging temporal stability and native protein distribution in transient and stable expression studies ", Plant J. 64: 355-365), for which the cDNA of ⁇ i / TST1 was amplified and the stop codon was removed by PCR using the ⁇ TST1 primers containing the attB1 and attB2 sites cloned via a BP reaction into pDONRZEO (Invitrogen, Heidelberg, DE), followed by an LR reaction in pUBC-GFP-Dest.
• the pUBQ: ⁇ vTST2.1-GFP construct was prepared as follows: the entire open reading frame of the ⁇ TST2 .1 gene was made with the primers
• Nucleotide sequence with Xhol / Pstl excised from this construct and inserted into a corresponding open with Xhol and Pstl vector pUBN-nYFP-Dest, which mediates hygromycin resistance in transformed plants. Digestion of pUBN-nYFP-Dest with Xhol / Pstl resulted in a complete
• the Agrobacterium tumefaciens strain GV3101 was used as a carrier of the nucleotide sequence coding for the gene 19K and for the corresponding
• Bacterial cells were resuspended in 3 ml of Agromix and kept in the dark for 2 to 3 hours at 28 ° C. For infiltration, 1 ml of the 19K-containing Agrobacterium suspension was mixed with 1 ml of the Agrobacterium suspension containing pUBQ: ⁇ iTST1-GFP, pUBQ: TST2.1-GFP or pUBQ.GFP and mixed with 2 ml of Agromix.
• Example 8 Analysis of the membrane proteome of the vacuoles from cells of the
• the vacuoles of taproot cells were isolated from five-month-old beetroots (Beta vulgaris) of the variety "Belladonna KWS" and the vacuolar membrane was enriched by high-speed centrifugation.
• the hydrophobic membrane proteins were washed out with the acetone several times Tonoplast fraction precipitated, subsequently in one
• ßvTST2.1 The subcellular localization of ßvTST2.1 was investigated in stable with pUBQ: ßvTST2.1-GFP transformed ⁇ ftst1-2 double gene knockout mutants.
• a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, DE) was used for fluorescence microscopy. All pictures were taken with a Leica HCX PL APO 63x / 1.20w motCORR CS lens. The image was processed with the Leica Application-Suite Advanced Fluorescence Lite software.
• the subcellular localization of the protein was determined by using a ⁇ vTST2.1-GFP fusion protein was stably expressed in Arabidopsis.
• the variety "Belladonna KWS” is known as a sugar beet variety, which has a high sucrose concentration and already two months after planting out a sucrose concentration of about 160 pmol xg "1
• the taproots of the variety "Brigadier” contained less than 70 pmol of sucrose per fresh weight after two months of growth, and they only accumulated about 195 of sucrose per g of fresh weight in the following three months ( Figure 3).
• the amount of ßvTST2.1 mRNA in the taproots of the variety "Belladonna KWS” was about 2.6 times higher than in the taproots of the variety "Brigadier” (FIG. 4).
• the amount of ⁇ vTST2.1 mRNA did not change significantly in both species compared to the amount after 3 months of growth, so that even after a five month growth and development phase, the amount of mRNA for ßvTST2.1 in taproots of the Type "Belladonna KWS" still about 2.6 times higher than in the
• the mRNA level for ⁇ TST2.1 in the leaves was consistently lower than the mRNA level for Î2vTMT1, Î2VTMT2.2 and Î2vTMT3, while the mRNA level for Î2VTST2.1 in the taproot was always higher than that mRNA level for the other isoforms (Figure 6).
• Example 12 ßvTST2.1 -mediated Tonoplastentransport of sucrose
• Intact vacuoles of transformed protoplasts could be identified after mild hypoosmotic lysis by their green color.
• Sucrose gradient couples The latter function is a biochemical prerequisite for the sugar beet to accumulate high levels of sucrose in the vacuoles of its taproots.
• ßvTST2.1 does not allow the glucose-mediated export of protons.
• the isoform ß ⁇ TST1 mediates both a sucrose-related and a glucose-related one
• Example 13 Sucrose specificity of BvTST2.1 in vivo
• AfTMT double-gene knockout mutants lacking either of the two major mononuclear tonoplastoid transporter proteins were either PUBQ: ßvTST2.1-GFP construct or pUBQ: ßi / TST1 Transformed construct.
• the transformants grew in

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